Methods for inducing in vivo tolerance

ABSTRACT

The present invention encompasses methods for inducing in vivo tolerance to a foreign tissue.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the priority of U.S. provisional application No.61/184,901, filed Jun. 8, 2009, which is hereby incorporated byreference in its entirety.

GOVERNMENTAL RIGHTS

This invention was made with government support under T32CA009547 and1F32AI08006201A1 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The invention encompasses methods for inducing in vivo tolerance to aforeign tissue.

BACKGROUND OF THE INVENTION

Whereas transplantation technology of both solid organs andhematopoietic cells has the potential to cure a variety of disorders,there are still treatment-related mortalities associated with theseprocedures, including toxicities of chemoradiotherapy, infectiouscomplications, and Graft-versus-Host Disease (GVHD). These relatedmortalities restrict the application of transplantation technology.

In GVHD, immune cells in a transplanted graft recognize the host asforeign, and mount an immune response to the host. GVHD can occur wheneither tissue or cells are transplanted (e.g. a solid organ orhematopoietic cells). Alternatively, a host can recognize thetransplanted tissue or cell as foreign. Hence, there is a need in theart for methods of inducing in vivo tolerance to a foreign tissue orcell in a subject.

SUMMARY OF THE INVENTION

One aspect of the present invention encompasses a method for treatingGVHD. The method typically comprises administering an anti-BTLA antibodyto a subject at substantially the same time the subject is exposed to agraft.

Another aspect of the present invention encompasses a method forinducing in vivo tolerance to a foreign tissue in a subject. The methodusually comprises administering to the subject an anti-BTLA antibody atsubstantially the same time as the foreign tissue exposure.

Other aspects and iterations of the invention are described morethoroughly below.

REFERENCE TO COLOR FIGURES

The application file contains at least one photograph executed in color.Copies of this patent application publication with color photographswill be provided by the Office upon request and payment of the necessaryfee.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts graphs and micrographs showing that anti-BTLA treatmentpermanently prevents graft-vs-host disease. CB6F1 mice were lethallyirradiated and received 2.0×10⁷ BMCs and 1.0×10⁷ splenocytes fromparental C57BL/6 BTLA+/+ (closed squares) or BTLA−/− (open squares)donors. Percent body weight (A) and clinical score (B) were measured forforty days post transplant. CB6F1 were lethally irradiated and received2.0×10⁷ BMCs and 1.0×10⁷ splenocytes from parental C57BL/6 BTLA+/+ miceand a single 200 μg injection intraperitoneally of the control antibodyPIP (open circles) or the antibody 6A6 (closed circles). Percent bodyweight (C) and clinical score (D) were measured for about forty dayspost transplant. Histopathology (E) of the colon 143 days after BMT ofanimals that had received a single injection of 6A6 (left panel) or thecontrol antibody PIP (right panel) on the day of BMT. Originalmagnification for histopathology was 4×. Error bars indicate positivestandard deviations for each time point. *Statistically significantdifferences versus both control groups (P<0.05).

FIG. 2 depicts a series of graphs showing BTLA expression by recipienttissue does not promote GVHD, but anti BTLA treatment prevents GVHD longterm. A & B represent the same experiment as in FIGS. 1C & D extendedfor 143 days. Body weight loss (A) and a clinical score (B) were used asa measure of GVHD in recipient mice after BMT. Error bars indicatepositive standard deviations for each time point. CB6F1 BTLA+/+ mice(closed squares) or CB6F1 BTLA−/− (open squares) were lethallyirradiated and received 2.0×10⁷ BMCs and 1.0×10⁷ splenocytes fromparental C57BL/6 BTLA−/− donors. Body weight loss (C) and a clinicalscore (D) were used as a measure of GVHD in recipient mice after BMT.

FIG. 3 depicts a series of graphs showing that anti BTLA treatmentexerts its affects at the time of BMT and independent from BTLA-HVEMinteractions. CB6F1 mice were lethally irradiated and received 2.0×10⁷BMCs and 1.0×10⁷ splenocytes from parental C57BL/6 BTLA+/+ mice togetherwith either a single 200 μg injection intraperitoneally of controlantibody (open circles) or 6A6 (closed circles) on the day of BMT or asingle 200 μg injection of 6A6 14 days after BMT (triangles). Bodyweight loss (A) and a clinical score (B) were used as a measure of GVHDin recipient mice after BMT. CB6F1 were lethally irradiated and received2.0×10⁷ BMCs and 1.0×10⁷ splenocytes from parental C57BL/6 HVEM−/− miceand a single 200 μg injection intraperitoneally of control antibody PIP(open circles) or 6A6 (closed circles). Body weight loss (C) and aclinical score (D) were used as a measure of GVHD in recipient miceafter BMT. Error bars indicate positive standard deviations for eachtime point. *Statistically significant differences versus both controlgroups (P<0.05).

FIG. 4 depicts a series of graphs showing that anti-BTLA antibody 6A6does not deplete lymphocytes. C57BL/6 mice received 5.0×10⁶ CFSE-labeledsplenocytes from B6.SJL mice together with a single 200 μg injectionintraperitoneally of either control antibody or anti-BTLA antibodies6A6, 6F7. After 2 days splenocytes were stained for CD4, CD8, CD19, andanti-hamster (for 6A6 and PIP). Shown are numbers of either all donorCFSE+ cells (A) or CD19+, CD8+, and CD4+ subsets of CFSE+ cells (B)recovered from mice that had received either control antibody (openbars), 6A6 (filled bars) or 6F7 (shaded bars). Data shown are mean±SEM(n=3). (C) CB6F1 (CD45.1−) mice were lethally irradiated and received5.0×10⁷ splenocytes from B6.SJL (CD45.1+) mice together with a single200 μg injection intraperitoneally of either 6A6 or PIP. After 7 dayssplenocytes were stained for CD45.1 and anti-hamster. Shown is ahistogram detecting bound antibody to lymphocytes read out byanti-hamster intensity within the 45.1+ donor cell population of micethat had either received 6A6 (bold line) or PIP (shaded fill).

FIG. 5 depicts a series of graphs showing that CD4 T cell accumulationis modestly affected by 6A6 treatment while CD4 and CD8 T cellproliferation is unperturbed. CB6F1 (CD45.1−) mice were lethallyirradiated and received 5.0×10⁷ CFSE-labeled splenocytes from B6.SJL(CD45.1+) mice together with a single 200 μg injection intraperitoneallyof either the antibody 6A6 or the control antibody PIP. After 3 (A) and7 (B) days splenocytes were stained for CD45.1, CD4, and CD8. Shown arenumbers of CD4+(left) or CD8+(right) cells of the 45.1+ donor cellpopulation recovered from mice that had received either 6A6 (open bars)or PIP (filled bars). Data shown are mean±SEM (n=3). (C) Histograms ofcell division history indicated by CFSE-intensity for CD4+ (left) orCD8+ (right) cells within the 45.1+ donor cell population on days 3(upper panel) and 7 (lower panel) of mice that had either received 6A6(bold line) or PIP (shaded fill).

FIG. 6 depicts a series of graphs showing that 6A6 treatment does notchange the cytokine production of donor CD4 T cells. CB6F1 (CD45.1-H-2Kd+) mice were lethally irradiated and received 2.0×10⁷ BMCs and 1.0×10⁷splenocytes from B6.SJL mice, and either control antibody or 6A6. 7 daysafter BMT splenocytes were harvested and stimulated with PMA/Ionomycinfor 4 hours. Following restimulation cells were stained for CD4, andeither IL-2, IFNγ, IL-17 and IL-4 or Isotype controls for the cytokines.(A) Plots show CD4+ cells and FSC, further gated on all CD4+ cells. Thetop two rows show isotype controls for control and 6A6 treated mice. Thebottom two rows show the production of the indicated cytokine followingeither control or 6A6 treatment. (B) Same experiment as in (A). Thegraph shows the percentage of CD4+ cells producing the indicatedcytokine.

FIG. 7 depicts a series of graphs showing that direct engagement of BTLAon donor CD4 T cells leads to an increased frequency of CD4+ FoxP3+cells. CB6F1 (CD45.1-H-2 Kd+) mice were lethally irradiated and receivedeither 2.0×10⁷ BMCs and 1.0×10⁷ B6.SJL BTLA+/+ splenocytes alone (B) ora 1:1 mixture of B6.SJL BTLA+/+ (CD45.1+H-2 Kd−) and C57BL/6 BTLA−/−(CD45.1-H-2 Kd−) donor cells (A and C) with either a single 200 μginjection intraperitoneally of control antibody or 6A6. After 7 dayssplenocytes were stained for CD45.1, H-2 Kd, CD4, and intracellularlyfor FoxP3. (A) Shown are plots for CD45.1 and H-2 Kd (left) and CD4 andFoxP3 (right) gated on C57BL/6 BTLA−/− (CD4+CD45.1− H-2 Kd−) or C57BL/6BTLA+/+ (CD4+CD45.1+ H-2 Kd−) donor cell populations as indicated.Numbers represent the percentage of cells within the indicated gates.(B) Shown are the percentage of CD45.1+CD4+FoxP3+ cells as a percentageof all CD45.1+CD4+B6.SJL BTLA+/+ derived donor cells. Data shown aremean±SEM (n=5). (C) Same experiment as in (A). Shown are the percentageof CD4+FoxP3+ cells as a percentage of all donor CD4+ cells from eitherB6.SJL BTLA+/+ mice (left) or from C57BL/6 BTLA−/− mice (right) thatreceived either control antibody (open bars) or 6A6 (filled bars). Datashown are mean±SEM (n=5)

FIG. 8 depicts a series of graphs showing that anti BTLA treatmentexpands pre-existing Tregs within the BMT. CB6F1 (CD45.2+H-2 Kd+) micewere lethally irradiated and received 2.0×10⁷ BMCs and 1.0×10⁷ WT B6.SJL(CD45.1+H-2 Kd−) splenocytes along with 1×10⁶ purified CD4+FoxP3− Tcells from B6.FoxP3GFP mice (CD45.2+H-2 Kd−) with either a single 200 uginjection intraperitoneally of control antibody or 6A6. After 7 dayssplenocytes were stained for CD45.1, CD45.2, H-2 Kd, CD4, and eitherintracellularly for FoxP3, or FoxP3 expression was determined by GFPexpression. (A) Donor cells are identified by lacking H2-Kd andexpressing CD4, then expression of CD45.2 or CD45.1 is used to determinethe origin of the donor. Expression of intracellular FoxP3 within CD4 Tcells from the WT donor (CD5.1+) is shown. Expression of FoxP3 asreported by GFP from the B6.FoxP3GFP donor (CD45.2+) is shown. (B) Sameexperiment as in (A). Shown are the percentage of CD4+FoxP3+ cells as apercentage of all donor CD4+ cells from either WT donors (left) or fromB6.FoxP3GFP donors (right) that received either control antibody (openbars) or 6A6 (filled bars). Data shown are mean±SEM (n=5)

FIG. 9 depicts a series of graphs showing that 6A6 does not expandsteady state Tregs. B6.FoxP3GFP mice were injected with 200 ug of eithercontrol antibody or 6A6 intraperitoneally and splenocytes were harvested6 days later. Cells were stained with CD4 and FoxP3 expression wasdetermined by GFP expression. (A) Plots showing the percentage of allcells that express CD4 and FoxP3 following control antibody (right) or6A6 treatment (left). (B) Same experiment as in (A) showing the percentof FoxP3+ cells within CD4+ T cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of inducing in vivo tolerance toforeign tissue. In certain embodiments, a method of the invention mayinduce in vivo tolerance without causing substantial systemic immunesuppression in the host. Such a method may be used, in part, to preventand/or treat GVHD. Generally speaking, the method comprisesadministering an anti-BTLA antibody to a subject exposed to foreigntissue. Typically, the anti-BTLA antibody administration occurs atsubstantially the same time as the foreign tissue exposure. In exemplaryembodiments, only one dose of an antibody is administered.

I. Methods for Inducing In Vivo Tolerance

The present invention encompasses a method for inducing in vivotolerance to foreign tissue. The method typically comprisesadministering an anti-BTLA antibody to a subject at substantially thesame time the subject is exposed to the foreign tissue. As used herein,“in vivo tolerance” refers to the substantial lack of immune responsespecific for the foreign tissue. The immune response may stem from therecipient subject mounting an immune response to a foreign tissue, orconversely, the immune response may stem from the foreign tissuemounting an immune response to the recipient subject (e.g. GVHD).Methods of measuring in vivo tolerance are commonly known in the art.

Suitable subjects have been, or will be, exposed to a foreign tissue.The term “foreign tissue,” as used herein, may encompass a bone marrowtransplant, an organ transplant, a blood transfusion, or any otherforeign tissue or cell that is purposefully introduced into a subject.

In certain embodiments, a method of the invention may be used to inducein vivo tolerance without causing substantial systemic immunesuppression in the host. Methods of detecting substantial systemicimmune suppression are known in the art, and may include, for example,measuring and detecting the ability of the host immune cells to respondto a stimulus in vitro.

In one embodiment, the present invention encompasses a method fortreating graft v. host disease (GVHD). Generally speaking, a method ofthe invention typically comprises administering an anti-BTLA antibody toa subject at risk for GVHD at substantially the same time the subject isexposed to the graft. A subject at risk for GVHD, generally speaking, isa subject exposed to a graft comprising viable and functional immunecells, where the graft is not 100% histocompatible with the subject. Insome embodiments, the subject is immunocompromised. In an exemplaryembodiment, the subject is human.

As used herein, “treating” refers to preventing GVHD or amelioratingGVHD symptoms in a subject. For instance, in one embodiment, treatingGVHD refers to substantially preventing GVHD associated weight loss. Inanother embodiment, treating GVHD means that there is no detectablecellular infiltrate in a target organ after the host is exposed to theforeign tissue. Suitable target organs in the context of GVHD mayinclude the liver, skin and mucosa, the gastrointestinal tract, the bonemarrow, the thymus, and the lungs.

In yet another embodiment, treating GVHD refers to decreasing theclinical score of the subject. For instance, in mice evidence of GVHDmay be scored by assessing five clinical parameters: weight loss,posture (hunching), activity, fur texture, and skin integrity.Individual mice receive a score of 0 to 2 for each criteria (maximumscore of 10). See Table A below.

TABLE A Criteria Grade 0 Grade 1 Grade 2 Weight loss <10% >10% to<25% >25% Posture Normal Hunching noted Severe hunching Activity NormalMild to moderately Stationary unless decreased stimulated Fur textureNormal Mild to moderate Severe ruffling/ ruffling poor grooming SkinNormal Scaling of paws/tail Obvious areas of integrity denuded skinAnalogously, in humans clinical scoring of acute GVHD may be performedby assessing three parameters (skin findings, liver findings (Bilirubinlevel, mg/dL), and gut findings) using the staging highlighted in TableB below. Overall clinical scoring of acute GVHD in humans may then becalculated using Table C below:

TABLE B Liver Findings (Bilirubin level, Stage Skin Findings mg/dL) GutFindings + Maculopapular 2-3 Diarrhea 500-1000 mL/d rash on <25% orpersistent nausea of body surface ++ Maculopapular 3-6 Diarrhea1000-1500 mL/d rash on 25-50% of body surface +++ Generalized  6-15Diarrhea >1500 mL/d erythroderma ++++ Desquamation >15 Pain with or andbullae without ileus

TABLE C Stage Functional Overall Grade Skin Liver Gut Impairment 0(None) 0 0 0 0 I (Mild) + to ++ 0 0 0 II (Moderate) + to +++ + + + III(Severe) ++ to +++ ++ to +++ ++ to +++ ++ IV (Life- ++ to ++++ ++ to++++ ++ to ++++ +++ threatening)

In some embodiments, a method of the invention may comprise decreasingthe clinical score of a subject, from, for example, a IV to a III, a IIIto a II, a II to a I, or a I to a 0 when the subject is human. In otherembodiments, a method of the invention may comprise decreasing theclinical score of a subject, from, for example, 10 to 9, 9 to 8, 8 to 7,7 to 6, 6 to 5, 5 to 4, 4 to 3, 3 to 2, 2 to 1, or 1 to 0 when thesubject is a mouse.

Generally speaking, if a method of the invention is used to decrease theclinical GVHD score of a subject, the decrease may be calculated withrespect to either A) the difference between a first score calculatedwithin 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 days of the foreign tissueexposure and a second score calculated after treatment of the subjectwith an anti-BTLA antibody, or B) the difference between a first scorecalculated within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 days of the foreigntissue exposure (in a subject treated with anti-BTLA antibody) and atypical baseline score of a control subject exposed to a foreign tissuewithout anti-BTLA antibody treatment.

(a) anti-BTLA Antibody

A method of the invention encompasses administering an anti-BTLAantibody. Generally speaking, the anti-BTLA antibody is capable ofbinding to BTLA and initiating the expansion of a pre-existing pool ofTreg cells. In some embodiments, the anti-BTLA antibody does not depleteT cells expressing BTLA. In other embodiments, the anti-BTLA antibodydoes not fix complement. In certain embodiments, the anti-BTLA antibodyrecognizes the same epitope as the anti-BTLA antibody 6A6. The 6A6anti-BTLA antibody is commonly known in the art. In certain otherembodiments, the anti-BTLA antibody recognizes amino acid epitopes onthe surface of BTLA that interacts with HVEM. This surface is generallyconserved in humans and mice. (See J Biol Chem. 2005 Nov. 25;280(47):39553-61 and J. Immunol. 2008 Jan. 15; 180(2):940-7, each ofwhich is hereby incorporated by reference in their entirety). Forinstance, the anti-BTLA antibody may recognize the Q27, C49, and Q66amino acids of mouse BTLA. In each of the above embodiments, theconstant region of the antibody may be human.

An antibody of the invention may be generated using BTLA, or a fragmentthereof, as an immunogen using methods that are well known in the art.Identification and selection of an antibody that binds to BTLA may beperformed using methods commonly known in the art. For more details, seethe Examples.

Usually, an anti-BTLA antibody of the invention is a monoclonalantibody. Monoclonal antibodies that bind to BTLA may be prepared usinga technique that provides for the production of antibody molecules bycontinuous cell lines in culture. These include, but are not limited to,the hybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique. (See, e.g., Kohler, G. et al. (1975) Nature256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:3142; Cote,R. J. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030; and Cole,S. P. et al. (1984) Mol. Cell Biol. 62:109-120.)

In addition, techniques developed for the production of “chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity may be used. (See, e.g., Morrison, S. L. et al.(1984) Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M. S. et al.(1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature314:452-45). Alternatively, techniques described for the production ofsingle chain antibodies may be adapted, using methods known in the art,to produce single chain antibodies that bind to BTLA. Antibodies withrelated specificity, but of distinct idiotypic composition, may begenerated by chain shuffling from random combinatorial immunoglobulinlibraries. (See, e.g., Burton, D. R. (1991) Proc. Natl. Acad. Sci. USA88:10134-10137.)

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature.(See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299.)

Antibody fragments that contain specific binding sites for BTLA orfragments thereof may also be generated. For example, such fragmentsinclude, but are not limited to, F(ab′)2 fragments produced by pepsindigestion of the antibody molecule and Fab fragments generated byreducing the disulfide bridges of the F(ab′)2 fragments. Alternatively,Fab expression libraries may be constructed to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity.(See, e.g., Huse, W. D. et al. (1989) Science 246:1275-1281.)

In the production of antibodies, screening for the desired antibody maybe accomplished by techniques known in the art, e.g. ELISA(enzyme-linked immunosorbent assay). Various immunoassays may be usedfor screening to identify antibodies having the desired specificity.Numerous protocols for competitive binding or immunoradiometric assaysusing either polyclonal or monoclonal antibodies with establishedspecificities are well known in the art. Such immunoassays typicallyinvolve the measurement of complex formation between BTLA and itsspecific antibody.

In some embodiments of the invention, an antibody may be conjugated to acomplex, such as a therapeutic complex or an imagining complex. Methodsof conjugating antibodies to various complexes are known in the art. Inother embodiments, an antibody of the invention may be labeled with adetectable marker. The marker may be either non-covalently or covalentlyjoined to an antibody of the present invention by methods generallyknown in the art. Detectable markers suitable for use in the inventiongenerally comprise a reporter molecule or enzyme that is capable ofgenerating a measurable signal. By way of non-limiting example, suchdetectable markers may include a chemiluminescent moiety, an enzymaticmoiety (e.g. horse-radish peroxidase), a fluorescent moiety (e.g. FITC)or a radioactive moiety. Additionally, in some embodiments, an antibodyof the invention may be labeled with avidin or biotin.

(b) Administration

Generally speaking, an anti-BTLA antibody is administered atsubstantially the same time the subject is exposed to the foreigntissue. As used herein, “substantially the same time” means that theantibody is administered close enough to the foreign tissue exposure toachieve a suppressive environment of alloreactive T cells. In oneembodiment, the anti-BTLA antibody is administered at the same time asthe foreign tissue exposure. In another embodiment, the anti-BTLAantibody is administered before the foreign tissue exposure. In yetanother embodiment, the anti-BTLA antibody is administered after foreigntissue exposure. In exemplary embodiments, the antibody is administeredclose enough to the foreign tissue exposure to treat GVHD. In otherexemplary embodiments, the antibody is administered close enough to theforeign tissue exposure to induce in vivo tolerance.

An anti-BTLA antibody may be administered to the subject once, or morethan once. For instance, an anti-BTLA antibody may be administered 1, 2,3, 4, 5, 6, 7, 8, 9, 10, or more than 10 times. By way of non-limitingexample, an anti-BTLA antibody may be administered at the time of theforeign tissue exposure, and after foreign tissue exposure.Alternatively, an anti-BTLA antibody may be administered before foreigntissue exposure and at the time of foreign tissue exposure. In anotheralternative, an anti-BTLA antibody may be administered before and afterforeign tissue exposure. In still another alternative, an anti-BTLAantibody may be administered before foreign tissue exposure, at the timeof foreign tissue exposure, and after foreign tissue exposure.

Generally speaking, in each of the above embodiments, an anti-BTLAantibody may be administered within 10 days of exposure to a foreigntissue. For instance, an anti-BTLA antibody may be administered 10, 9,8, 7, 6, 5, 4, 3, 2, or 1 days before foreign tissue exposure, and/or10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 days after foreign tissue exposure. Insome embodiments, an anti-BTLA antibody may be administered within aweek of exposure to a foreign tissue.

Usually, the amount of anti-BTLA antibody administered is between about5 μg/g body weight to about 20 μg/g body weight. In some embodiments,the amount of anti-BTLA antibody administered is about 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 μg/g body weight.

An antibody of the invention may be incorporated into a pharmaceuticalcomposition suitable for administration to a subject. Typically, thepharmaceutical composition comprises an antibody or antibody fragment ofthe invention and a pharmaceutically acceptable carrier. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible. Examples of pharmaceutically acceptablecarriers include one or more of water, saline, phosphate bufferedsaline, dextrose, glycerol, ethanol and the like, as well ascombinations thereof. In many cases, it will be preferable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Pharmaceuticallyacceptable carriers may further comprise minor amounts of auxiliarysubstances such as wetting or emulsifying agents, preservatives orbuffers, which enhance the shelf life or effectiveness of the antibodyor antibody portion.

The pharmaceutical compositions of this invention may be in a variety offorms. These include, for example, liquid, semi-solid and solid dosageforms, such as liquid solutions (e.g., injectable and infusiblesolutions), dispersions or suspensions, tablets, pills, powders,liposomes and suppositories. The preferred form depends on the intendedmode of administration and therapeutic application. Typical preferredcompositions are in the form of injectable or infusible solutions, suchas compositions similar to those used for passive immunization of humanswith other antibodies. The preferred mode of administration isparenteral (e.g., intravenous, subcutaneous, intraperitoneal,intramuscular). In a preferred embodiment, the antibody is administeredby intravenous infusion or injection. In another preferred embodiment,the antibody is administered by intramuscular or subcutaneous injection.

Pharmaceutical compositions may be sterile and are typically stableunder the conditions of manufacture and storage. The composition may beformulated as a solution, microemulsion, dispersion, liposome, or otherordered structure suitable to high drug concentration. Sterileinjectable solutions can be prepared by incorporating the activecompound (i.e., antibody or antibody fragment) in the required amount inan appropriate solvent with one or a combination of ingredientsenumerated above, as required, followed by filtered sterilization.Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle that contains a basic dispersion medium and therequired other ingredients from those detailed above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The proper fluidity of a solution may be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions may be achieved byincluding an agent that delays absorption, for example, monostearatesalts and gelatin, in the composition.

An antibody of the invention, or a pharmaceutical composition comprisingan antibody of the invention, may be administered to a subject. Anantibody of the present invention may be administered by a variety ofmethods known in the art, although for many therapeutic applications,the preferred route/mode of administration is intravenous injection orinfusion. As will be appreciated by the skilled artisan, the routeand/or mode of administration will vary depending upon the desiredresults. In certain embodiments, the active compound may be preparedwith a carrier that will protect the compound against rapid release,such as a controlled release formulation, including implants,transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers may be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In certain embodiments, an antibody of the invention may be orallyadministered, for example, with an inert diluent or an assimilableedible carrier. The composition (and other ingredients, if desired) mayalso be enclosed in a hard or soft shell gelatin capsule, compressedinto tablets, or incorporated directly into the subject's diet. For oraltherapeutic administration, the compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.To administer a compound of the invention by other than parenteraladministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples that follow representtechniques discovered by the inventors to function well in the practiceof the invention. Those of skill in the art should, however, in light ofthe present disclosure, appreciate that many changes can be made in thespecific embodiments that are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention, therefore all matter set forth or shown in the accompanyingdrawings is to be interpreted as illustrative and not in a limitingsense.

EXAMPLES

The following examples illustrate various iterations of the invention.

Introduction

Allogeneic replacement of an abnormal or malignant hematopoietic systemby allogeneic hematopoietic stem-cell transplantation (aHSCT) from ahealthy donor can cure a variety of blood cell disorders and inducelong-lasting tolerance to foreign tissues (1-3). The hightreatment-related mortality of this procedure due to toxicities ofchemoradiotherapy, infectious complications, and Graft-versus-HostDisease (GVHD), however, restricts its widespread application. Thus itis necessary to develop new methods for inhibiting GVHD, whilemaintaining the positive effects of donor T cells for the broaderapplication of aHSCT in patients with inherited disorders ofhematopoiesis (such as sickle cell anemia or aplastic anemia) orpatients without an MHC-matched donor. The regulation of multiplecostimulatory molecules on donor-derived T cells is crucial to theoutcome of GVHD. Stimulation of co-inhibitory molecules amelioratesGVHD, while co-stimulatory molecules lead to the pathogenesis of GVHD.In addition to the CD28 family of co-stimulatory molecules (4-12),functional roles for the TNF receptor family of co-stimulatory ligandsand receptors has been implicated in the pathogenesis of GVHD (13-20).Here it is shown that a single administration of a non-depletingmonoclonal antibody directed against BTLA (22) after aHSCT leads to theexpansion of regulatory T cells and the permanent prevention of GVHD.Thus, targeting BTLA could improve the safety of aHSCT and broaden itsapplication to common non-life threatening diseases. In addition,targeting BTLA may induce tolerance to solid organ grafts.

Example 1 BTLA is not normally engaged during GVHD

To determine whether BTLA signaling played a functional role in thedevelopment GVHD, wild type and BTLA-deficient mice (23) were firstexamined using a parental into irradiated F1 model. In this model GVHDmanifests as a result of a partial MHC mismatch achieved throughparental donor cells of the H-2^(b) haplotype and the lethallyirradiated recipients of the H-2^(b/d) haplotype. Bone marrow andsplenocytes from either wildtype or BTLA−/− were transferred intolethally irradiated CB6F1 recipients (FIG. 1A). As expected,transplantation of wild type donor cells into CB6F1 recipients caused adecrease in body weight of approximately 30%, and generated clinicalscores (24) of about 3 which persisted for greater than 40 days (FIG.1B). BTLA−/− donor cells caused GVHD to a similar magnitude as wild typedonor cells (FIGS. 1A & B).

To determine if recipient expression of BTLA played a role in GVHD wetransferred either wildtype or BTLA−/− bone marrow and splenocytes intolethally irradiated BTLA−/− CB6F1 hosts (FIG. 2). Donor cells from bothwild type and BTLA−/− caused similar disease in the BTLA−/− hosts, asthey displayed similar weight loss and GVHD clinical scores as comparedto BTLA+/+ recipients (FIG. 1). Collectively these data suggest thatBTLA is not normally engaged by either donor or recipient cells duringthe pathophysiological process of developing GVHD.

Example 2 Anti-BTLA Antibody Prevent GVHD

Since BTLA provides inhibitory signaling (23, 25, 26) and is activeduring several immunoregulatory models (27) (including infectiousdiseases such as malaria (28)), we tested whether direct engagement ofBTLA during bone marrow transplantation could regulate the developmentof GVHD. To test this hypothesis, we used an anti-BTLA monoclonalantibody, 6A6 (22; herein incorporated by reference in its entirety),delivered at the time of bone marrow transplantation (FIG. 1B). As acontrol, an irrelevant antibody that recognizes bacterial GST (PIP) wasused (29). Treatment with the control Ab had no effect on GVHDprogression, and mice receiving control antibody showed progression ofdisease similar to that of mice receiving wild type donor cells withouttreatment (FIG. 1A). Furthermore, mice receiving control antibody showedpersistent GVHD up to 140 days, with clinical scores between 3 and 4 forthis entire period (FIGS. 2A & B). In contrast, transplantation of wildtype donor cells with a single treatment using 200 ug of 6A6 led to acomplete prevention of GVHD with initial weight loss less than 10%, andno clinical signs of disease (FIGS. 1C & D). This treatment led to longterm prevention of GVHD as no signs of clinical disease developed at anytime up to 140 days after BMT (FIG. 2).

The colon is a major target organ of GVHD in this model (30) andrecipient mice treated with the control antibody developed typical signsof GVHD, with massive thickening of the lamina propria, muscular layersand intense inflammatory infiltrates and ulceration (FIG. 1E leftpanel). By contrast, histological analysis of the colon from mice thatreceived 6A6 treatment revealed essentially normal architecture with nocellular infiltrates into the lamina propria or epithelium. These datasuggest a single administration of anti-BTLA antibody prevents GVHD longterm as defined by no weight loss, low clinical GVHD score and nodetectable cellular infiltrate in a target organ such as the colon.

Example 3 Substantially Delayed Antibody Administration does not PreventGVHD

Since the single administration of 6A6 permanently prevented thedevelopment of GVHD in this system, it was tested whether BTLA treatmentby 6A6 could either reverse or prevent disease if administered at asubstantially later point in time. Therefore the experiments wererepeated using administration of 6A6 at day 14 following BMT (FIG. 3A).Administration of 6A6 at the time of transplantation did preventdisease, as expected, however substantially delayed administration of6A6 did not cure GVHD. There was no statistical difference between theweight loss or the clinical score between control mice that received noantibody, or mice that received 6A6 on day 14 after BMT, suggesting thatsubstantially delayed administration has no effect on preventing GVHD.

Example 4 Antibody Mechanism is Independent of HVEM Interaction

Because BTLA binds to HVEM (31, 32), a member of the TNF receptorfamily, it was possible that the prevention of GVHD seen after 6A6administration was due to interference with BTLA and HVEM interactionsin the host. If this were true, then 6A6 should have no curative effectswhen HVEM−/− donor cells were transplanted. HVEM−/− donor cells causeGVHD, when the control antibody PIP was administered (FIG. 3D). However,GVHD caused by HVEM−/− donor cells is prevented by treatment with 6A6 atthe time of transplantation. Interestingly, the degree of weight lossand the clinical scores are somewhat reduced when HVEM−/− donor cells(FIG. 3D) induce GVHD compared to wild type donor cells (FIG. 1B),consistent with a recent report that HVEM and LIGHT act asco-stimulatory molecules promoting pathophysiology in GVHD (16). Theseresults demonstrate that the curative effects of 6A6 on GVHD operate ina manner that is independent of HVEM, consistent with an active signalthrough BTLA on donor cells.

Example 5 Antibody does not Deplete T Cells

6A6 treatment leads to a drastic prevention in GVHD, therefore it wasdetermined whether 6A6 exerted its effects simply by depletion of donorT cells that expressed BTLA. Wildtype donor cells were labeled with CFSEand transferred into wildtype recipients. At the time of transfer micewere treated with control antibody PIP, anti-BTLA 6A6 or anti-BTLA 6F7.Two days after transfer similar numbers of total CFSE+ cells, as well aslymphocytes CD19+, CD4+ and CD8+ cells were recovered from mice thatreceived either control antibody PIP or anti-BTLA 6A6 (FIG. 4A). Incontrast, anti-BTLA antibody 6F7 lead to a depletion of lymphocytes,most drastically CD19+ cells as they express the highest levels of BTLA.Furthermore, we were able to detect 6A6 bound to donor derived cells 7days after transfer where various levels of bound 6A6 is detected and islikely the demonstration of BTLA high cellular expression of BTLA on Bcells compared to T cells (FIG. 4B). Thus 6A6 is not a depletingantibody. This is likely because it is an IgG1 hamster monoclonalantibody and is unlikely to fix mouse compliment, whereas anti-BTLAantibody 6F7 mediates significant lymphocyte depletion because it is aIgGK mouse monoclonal antibody (22).

Example 6 Antibody Mechanism is via Treg Population

BTLA signaling in T cells generally provides an inhibitory signal (23,25, 26), however the precise mechanisms of this inhibition, themolecules involved, and the targets of inhibition are still somewhatobscure (25, 33, 34). It has been shown that BTLA engagement with HVEMleads to a decrease in cell proliferation in vitro (31). In additionBTLA deficient T cells are less likely to become anergic (35). Thereforeit was sought to determine whether 6A6 engagement results in a decreasein proliferation or IL-2 production by T cells. We CFSE labeled donorsplenocytes and transferred them into lethally irradiated CB6F1recipients and treated with either control or 6A6 antibody to asses theproliferation rate following BMT. CD4 and CD8 T cell proliferation 3 and7 days after transfer was similar between mice that received controlantibody and 6A6. This was demonstrated by similar cell division historyprofiles with CFSE dilution (FIG. 5C). The cellular expansion of donorCD8 T cells in mice treated with 6A6 resulted in a 50% reduction of cellaccumulation 3 days after transplantation compared to control (FIG. 5A).However, 7 days after transplantation after robust proliferation (FIG.5C) the difference in cell accumulation between control and 6A6 treatedmice was no longer significant (FIG. 5B).

CD4 T cell accumulation was reduced by approximately 70% on day 3 inmice treated with 6A6 (FIG. 5B), this decreased expansion in CD4 T cellspersisted at day 7 with approximately a 50% reduction in the expansionof donor derived T cells in mice treated with 6A6 compared to PIP.Furthermore, to assay whether 6A6 treatment lead to an anergic phenotypeof CD4 T cells, we measured IL-2 production from cells 7 days posttransplantation. CD4 T cells from mice that received 6A6 treatmentproduced IL-2 similar to control treated mice (FIG. 6). These dataindicate that 6A6 treatment does not induce anergy in CD4 T cells asthey proliferate and produce IL-2. Furthermore, CD8 T cell expansion wasunaffected with 6A6 treatment by day 7 post transplantation and CD4 Tcell accumulation was only modestly affected.

GVHD is driven by a strong TH1 immune response and we sought todetermine if treatment with 6A6 causes a skewing in the cytokine profileof CD4 T cells 7 days after BMT. Mice that were treated with controlantibody produced mainly IFNγ, in addition, little IL-17 and IL-4 wasproduced by CD4 T cells which is the cytokine profile indicative of astrong TH1 response (FIG. 6). CD4 T cells from mice treated with 6A6antibody displayed the same strong TH1 cytokine profile as controltreated mice with slightly reduced IFNγ and IL-2 production (FIG. 6).The decrease in IFNγ production is possibly due to the curative affectof 6A6, yet no obvious skewing of the immune response occurred with noincrease in IL-17 or IL-4 production.

The only indication there is a difference in response when mice aretreated with 6A6 is the slight reduction in proliferation and productionof IL-2 by CD4 T cells. This phenotype is reminiscent of CD4+ Tregulatory cells which selectively express the transcription factorforkhead box P3 (Foxp3) (36). Recently a significant role for regulatoryT cells has been described for preventing GVHD in a manner similar toour observations following 6A6 treatment (37-40). To determine whetheranti BTLA antibody treatment was preventing GVHD through modulation of Tregulatory cells, we measured the expression of FoxP3 in CD4+ donor Tcells 7 days after bone marrow transplantation (FIG. 7A). Mice treatedwith the control antibody PIP, which developed GVHD, had 12%±3% of donorCD4 T cells which expressed Foxp3 (FIG. 7B). By comparison, when 6A6 wasused, there was a marked increase in the expression of Foxp3 (40%±5%) bydonor derived CD4 T cells (FIG. 7B). Thus the increased frequency ofTregs 7 days following anti-BTLA treatment is in agreement with themodest reduction of accumulation and IL-2 production of CD4 T cells. AsTregs are potent suppressors of immune function it is likely theselective expansion of Tregs following 6A6 treatment that results in theprevention of GVHD.

BTLA is expressed by many cells of the hematopoietic system, thus it wasnot clear if 6A6 engagement on T cells was necessary for the increase inFoxP3 or if another cell such as an APC received a signal from 6A6 whichresulted in priming of a CD4 T cell to become Tregs. Therefore weperformed a mixed BMT with wildtype and BTLA−/− bone marrow andsplenocytes. If 6A6 treatment directly induces Tregs there would be anincrease in Tregs from the wildtype donors only, in contrast indirectinduction of Tregs by an APC would result in an increase of Tregs fromboth wildtype and BTLA−/− donors. The increase in Tregs was observed inwildtype cells that were treated with 6A6 (FIGS. 7A & C). In contrast,cells from the BTLA−/− donor had no increase in Tregs following 6A6treatment compared to mice receiving control antibody (FIGS. 7A & C).Therefore 6A6 administration at the time of BMT directly engages BTLA onCD4 T cells to preferentially increase the frequency of Tregs.

Example 7 Antibody Mechanism Expands Existing CD4 Treg Population

The expansion of Tregs following 6A6 treatment could be the result ofeither the expansion of pre-existing Tregs in the splenic portion of theBMT or through peripheral conversion of naïve CD4 T cells into FoxP3+Tregs (41). To ascertain whether 6A6 could induce peripheral conversionof naïve CD4 T cells into Tregs we added FoxP3-negative CD4+ cells fromB6.FoxP3-GFP mice (42) and mixed them with the normal BMT. Treatmentwith control antibody increased the frequency of donor CD4+FoxP3+ cellsin the wildtype unpurified splenocyte fraction of the BMT as previouslyobserved (FIGS. 8A & B). However, the population of donor CD4 T cellsobtained from B6.FoxP3GFPKI which originally were CD4+FoxP3− failed toincrease their expression of FoxP3 as indicated by GFP expression 7 daysfollowing 6A6 treatment (FIGS. 8A & B). The expansion of pre-existingTregs likely requires T cell activation because 6A6 treatment directlyinto otherwise unmanipulated B6.FoxP3GFPKI mice did not preferentiallyincrease the frequency of CD4+FoxP3+ cells 6 days following whencompared to control mice (FIG. 9). In total, these results indicate thata single administration of 6A6 at the time of bone marrowtransplantation leads to the complete prevention of GVHD and theconcomitant expansion of pre-existing CD4 T regulatory cells that aredonor derived.

This study has demonstrated that although the natural progression ofGVHD does not normally engage BTLA in the parental into F1 irradiatedmodel, direct BTLA engagement using a non-depleting anti-BTLA antibodycan permanently prevent GVHD and increase the frequency of pre-existingdonor derived regulatory T cells. Once GVHD has been establishedanti-BTLA treatment had no effect suggesting early expansion of Tregs isimportant for achieving a suppressive environment of alloreactive donorT cells.

Material and Methods

Mice and bone marrow transplantation B6.SJL-Ptprca Pep3b/BoyJ (B6.SJL),C57BL/6, and C57BL/6×BALB/c F1 (CB6F1) mice were obtained from TheJackson Laboratory (Bar Harbor, Me.) or bred in our facility. BTLA^(−/−)(23), Hvem^(−/−) (43), and FoxP3GFP (42) mice were backcrossed toC57BL/6 for at least nine generations. Mice were 12-18 weeks old andfemale. All mice were kept under special pathogen-free conditions.

Cell transplantation and assessment of GVHD Mice received transplantsaccording to a standard protocol as previously described (30). Briefly,bone marrow cells were harvested by flushing tibia and femurs of donormice. For GVHD induction, CB6F1 (H-2^(b/d)) recipients were lethallyirradiated with 9 Gy total body irradiation (TBI) using a 137Cs sourceat a dose rate of ˜70 cGy/minute and reconstituted with bone marrowcells (BMCs) and additional splenocytes (2×10⁷ BMCs and 1×10⁷splenocytes) from indicated donors (H-2^(d)). GVHD was monitored bycalculating the loss in total body weight. Body weights were measuredbefore transplantation and 3 times a week after transplantation.Clinical GVHD intensity was scored by assessing weight loss, posture,activity, fur texture, and skin integrity (24). Histopathologic analysesof the bowel were performed on hematoxylin and eosin (H&E)—stainedtissue. Microscopic analyses were performed with a BX51 light microscope(Olympus, Hamburg, Germany) equipped with a 40×/0.75 NA objective lensand a DP70 camera (Olympus) using Cell A Analysis software (OlympusSoftware Imaging Solutions 1986-2007, Muenster, Germany). Experimentswere performed in accordance with national and institutional guidelines.

CFSE labeling and Flow Cytometery Cells were labeled with CFSE(carboxyfluorescein diacetate succinimidyl diester; Sigma-Aldrich) bybeing incubated for 8 min at 25° C. with 1 μM CFSE at a density of40×10⁶ cells per ml in PBS. Cells were incubated for 1 min with an equalvolume of FCS and were washed twice with media containing 10% (vol/vol)FCS. 50×10⁶ total cells were injected IV per mouse. Single cellsuspensions from spleens were analyzed by flow cytometry using thefollowing antibodies for detection: Kd-FITC (SF1-1.1), CD4-PECy7 and APC(RM4-5), anti-Armenian and Syrian hamster IgG cocktail-PE, CD19-APC(1D3) purchased from BD Pharmingen. Additional antibodies purchased fromeBioscience were also used: CD45.1-PECy7 and APC (A20), CD8-APCAlexaFluor 750 (53-6.7), CD4-APC AlexaFluor 750 (RM4-5). IntracellularFoxP3 was detected using eBioscience Mouse Regulatory T cell stainingKit with FoxP3-PE or APC (FJK-16s). For intracellular cytokine stainingsplenocytes were first restimulated with PMA/ionomycin for 4 hours andwere stained with antibodies to surface markers followed by fixationwith 2% formaldehyde for 15 minutes at room temperature. Cells were thenwashed once in 0.05% saponin and stained with anti-cytokine antibodies(anti IL-17 FITC, IL-2 PE, IFNg PE-Cy7 and IL-4 APC in 0.5% saponin. Allflow cytometry data were collected on a FACSCanto II (BD Biosciences)and were analyzed with FlowJo software (Tree Star).

Administration of antibody In some experiments mice received a singleintraperitoneal injection of 10-20 μg/g body weight of the IgG1 hamsterinjection of monoclonal anti-BTLA antibody 6A6, the IgGK mousemonoclonal anti-BTLA antibody 6F7 (Hurchla, 2005) or the hamstermonoclonal anti-GST antibody PIP (Gronowski, 1999) at indicated timepoints.

Statistical analysis A Student's unpaired two-tailed t-test was used forstatistical analyses of body weight data. Differences with P values of0.05 or less are considered significant.

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1. A method for treating GVHD, the method comprising administering anagonistic anti-BTLA antibody that does not deplete T cells that expressBTLA to a subject at substantially the same time the subject is exposedto a graft.
 2. The method of claim 1, wherein the antibody isadministered at the same time as the graft exposure.
 3. The method ofclaim 1, wherein the antibody is administered before the graft exposure.4. The method of claim 1, wherein the antibody is administered withinone week of the graft exposure.
 5. The method of claim 1, wherein theantibody expands a pre-existing Treg cell population.
 6. The method ofclaim 1, wherein the antibody is administered once.
 7. The method ofclaim 6, wherein the antibody is administered once, and at the time ofgraft exposure.
 8. A method for inducing in vivo tolerance to a foreignbone marrow in a subject, the method comprising administering anagonistic anti-BTLA antibody to a subject undergoing a bone marrowtransplant at substantially the same time as the foreign tissueexposure.
 9. The method of claim 8, wherein the immune system of thesubject is not systemically suppressed.
 10. The method of claim 8,wherein the antibody does not deplete T cells.
 11. The method of claim8, wherein the antibody is administered at the same time as the foreigntissue exposure.
 12. The method of claim 8, wherein the antibody isadministered before the foreign tissue exposure.
 13. The method of claim8, wherein the antibody is administered within one week of the foreigntissue exposure.
 14. The method of claim 8, wherein the antibody expandsa pre-existing Treg cell population.
 15. The method of claim 8, whereinthe antibody is administered once.
 16. The method of claim 15, whereinthe antibody is administered once, and at the time of foreign tissueexposure.
 17. A method for treating GVHD, the method comprisingadministering an agonistic anti-BTLA antibody to a subject undergoing abone marrow transplant at substantially the same time the subject isexposed to a graft.
 18. The method of claim 17, wherein the antibodydoes not deplete T cells.
 19. The method of claim 17, wherein theantibody is administered at the same time as the graft exposure.
 20. Themethod of claim 17, wherein the antibody is administered before thegraft exposure.
 21. The method of claim 17, wherein the antibody isadministered within one week of the graft exposure.
 22. The method ofclaim 17, wherein the antibody expands a pre-existing Treg cellpopulation.
 23. The method of claim 17, wherein the antibody isadministered once.
 24. The method of claim 23, wherein the antibody isadministered once, and at the time of graft exposure.
 25. A method fortreating GVHD, the method comprising administering a single dose of anagonistic anti-BTLA antibody to a subject at substantially the same timethe subject is exposed to a graft.
 26. The method of claim 25, whereinthe antibody does not deplete T cells.
 27. The method of claim 25,wherein the antibody is administered at the same time as the graftexposure.
 28. The method of claim 25, wherein the antibody isadministered before the graft exposure.
 29. The method of claim 25,wherein the antibody is administered within one week of the graftexposure.